PROJECT SUMMARY/ABSTRACT The human immune response to HIV-1 is complex with the virus demonstrating remarkable evasion mechanisms. Significant progress has recently been made in HIV vaccine development based on early-stage clinical trials and subsequent investigations into the nature of protective immunity. Several promising messenger RNA (mRNA)-based HIV vaccines that target the HIV envelope protein (Env) have been developed and tested with promising results in animal and nonhuman primate models. The vaccines are based on lipid nanoparticle (LNP) technology which relies on the LNP lipid bilayer to protect the mRNA cargo and promote cellular uptake but lacks the specificity to traffic the vaccine to dendritic cells (DC) and secondary lymphoid organs ? a deficiency addressed through depot, intradermal or subcutaneous injection. Rather than modifying the lipid bilayer of an mRNA-LNP vaccine to impart desirable physical and biological attributes, we propose a more versatile approach: decoupling vaccine functionality into layers that address extracellular and intracellular issues and goals separately. Toward this end, we propose to design and develop a customized, spherical DNA nanostructure (origami) construct to encapsulate an existing mRNA-LNP vaccine with the goals of increasing immunogenicity and prolonging specific antibody production. The origami will serve (1) as an anionic shell coated with ligands for DC targeting, (2) as an anchor for co-delivery of adjuvants, and (3) as a protective layer against enzyme degradation and nonspecific cellular interactions. We will develop and optimize protocols for synthesizing the origami-LNP complex and characterize it using atomic force microscopy, dynamic light scattering, fluorescence microscopy, and gel chromatography. We will then demonstrate the capacity of the resulting construct to transfect and effect expression of Env in cell cultures Env protein by western blot. Targeting and uptake will be assessed using labeled DNA constructs and fluorescence microscopy. Finally, we will evaluate vaccine efficacy to stimulate a specific humoral immune response in a murine model by quantifying antibody titers by ELISA assay. If successful, the proposed origami-LNP approach to nanocarrier design could shift current drug delivery practice not only for mRNA- based vaccines, but for a wide variety of therapeutic applications.